Guanylate cyclase receptor agonists for the treatment of tissue inflammation and carcinogenesis

ABSTRACT

A method of treatment of inflamed, pre-cancerous or cancerous tissue or polyps in a mammalian subject is disclosed. The treatment involves administration of a composition of at least one peptide agonist of a guanylate cyclase receptor and/or other small molecules that enhance intracellular production of cGMP. The at least one peptide agonist of a guanylate cyclase receptor may be administered either alone or in combination with an inhibitor of cGMP-dependent phosphodiesterase. The inhibitor may be a small molecule, peptide, protein or other compound that inhibits the degradation of cGMP. Without requiring a particular mechanism of action, this treatment may restore a healthy balance between proliferation and apoptosis in the subject&#39;s population of epithelial cells, and also suppress carcinogenesis. Thus, the method may be used to treat, inter alia, inflammation, including gastrointestinal inflammatory disorders, general organ inflammation and asthma, and carcinogenesis of the lung, gastrointestinal tract, bladder, testis, prostate and pancreas, or polyps.

RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 10/107,814, filed Mar. 28, 2002 now U.S. Pat. No. 7,041,786 andclaims the benefit of U.S. Patent Application No. 60/279,438, filed onMar. 29, 2001; U.S. Patent Application No. 60/279,437, filed on Mar. 29,2001; U.S. Patent Application No. 60/300,850, filed on Jun. 27, 2001;U.S. Patent Application No. 60/303,806, filed on Jul. 10, 2001; U.S.Patent Application No. 60/307,358, filed on Jul. 25, 2001; and U.S.Patent Application No. 60/348,646, filed on Jan. 17, 2002. The contentsof these applications are incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to the therapeutic use of guanylatecyclase receptor agonists as a means for enhancing the intracellularproduction of cGMP. The agonists may be used either alone or incombination with inhibitors of cGMP-specific phosphodiesterase toprevent or treat cancerous, pre-cancerous and metastatic growths,particularly in the gastrointestinal tract and lungs. In addition, theagonists may be used in the treatment of inflammatory disorders such asulcerative colitis and asthma.

BACKGROUND OF THE INVENTION

Uroguanylin, guanylin and bacterial ST peptides are structurally relatedpeptides that bind to a guanylate cyclase receptor and stimulateintracellular production of cyclic guanosine monophosphate (cGMP) (1-6).This results in the activation of the cystic fibrosis transmembraneconductance regulator (CFTR), an apical membrane channel for efflux ofchloride from enterocytes lining the intestinal tract (1-6). Activationof CFTR and the subsequent enhancement of transepithelial secretion ofchloride leads to stimulation of sodium and water secretion into theintestinal lumen. Therefore, by serving as paracrine regulators of CFTRactivity, cGMP receptor agonists regulate fluid and electrolytetransport in the GI tract (1-6; U.S. Pat. No. 5,489,670).

The process of epithelial renewal involves the proliferation, migration,differentiation, senescence, and eventual loss of GI cells in the lumen(7,8). The GI mucosa can be divided into three distinct zones based onthe proliferation index of epithelial cells. One of these zones, theproliferative zone, consists of undifferentiated stem cells responsiblefor providing a constant source of new cells. The stem cells migrateupward toward the lumen to which they are extruded. As they migrate, thecells lose their capacity to divide and become differentiated forcarrying out specialized functions of the GI mucosa (9). Renewal of GImucosa is very rapid with complete turnover occurring within a 24-48hour period (9). During this process mutated and unwanted cells arereplenished with new cells. Hence, homeostasis of the GI mucosa isregulated by continual maintenance of the balance between proliferationand apoptotic rates (8).

The rates of cell proliferation and apoptosis in the gut epithelium canbe increased or decreased in a wide variety of different circumstances,e.g., in response to physiological stimuli such as aging, inflammatorysignals, hormones, peptides, growth factors, chemicals and dietaryhabits. In addition, an enhanced proliferation rate is frequentlyassociated with a reduction in turnover time and an expansion of theproliferative zone (10). The proliferation index has been observed to bemuch higher in pathological cases of ulcerative colitis and other GIdisorders (11). Thus, intestinal hyperplasia is the major promoter ofgastrointestinal inflammation and carcinogenesis.

In addition to a role for uroguanylin and guanylin as modulators ofintestinal fluid and ion secretion, these peptides may also be involvedin the continual renewal of GI mucosa. Previously published data in WO01/25266 suggests a peptide with the active domain of uroguanylin mayfunction as an inhibitor of polyp development in the colon and mayconstitute a treatment of colon cancer. However, the mechanism by whichthis is claimed to occur is questionable in that WO 01/25266 teachesuroguanylin agonist peptides that bind specifically to a guanylatecyclase receptor, termed GC-C, that was first described as the receptorfor E. coli heat-stable enterotoxin (ST) (4). Knockout mice lacking thisguanylate cyclase receptor show resistance to ST in intestine, buteffects of uroguanylin and ST are not disturbed in the kidney in vivo(3). These results were further supported by the fact that membranedepolarization induced by guanylin was blocked by genistein, a tyrosinekinase inhibitor, whereas hyperpolarization induced by uroguanylin wasnot effected (12,13). Taken together these data suggest that uroguanylinalso binds to a currently unknown receptor, which is distinct from GC-C.

Other papers have reported that production of uroguanylin and guanylinis dramatically decreased in pre-cancerous colon polyps and tumortissues (14-17). In addition, genes for both uroguanylin and guanylinhave been shown to be localized to regions of the genome frequentlyassociated with loss of heterozygosity in human colon carcinoma (18-20).Taken together, these findings indicate that uroguanylin, guanylin andother peptides with similar activity may be used in the prevention ortreatment of abnormal colon growths. This proposal is bolstered by arecent study demonstrating oral administration of uroguanylin inhibitspolyp formation in mice (15,16).

Uroguanylin and guanylin peptides also appear to promote apoptosis bycontrolling cellular ion flux. Alterations in apoptosis have beenassociated with tumor progression to the metastatic phenotype. While aprimary gastrointestinal (GI) cancer is limited to the small intestine,colon, and rectum, it may metastasize and spread to such localities asbone, lymph nodes, liver, lung, peritoneum, ovaries, brain. By enhancingthe efflux of K⁺ and influx of Ca⁺⁺, uroguanylin and related peptidesmay promote the death of transformed cells and thereby inhibitmetastasis.

One of the clinical manifestations of reduced CFTR activity is theinflammation of airway passages (21). This effect may be due to CTFRregulating the expression of NF-KB, chemokines and cytokines (22-25).Recent reports have also suggested that the CFTR channel is involved inthe transport and maintenance of reduced glutathione, an antioxidantthat plays an important role in protecting against inflammation causedby oxidative stress (39). Enhancement of intracellular levels of cGMP byway of guanylate cyclase activation or by way of inhibition ofcGMP-specific phosphodiesterase would be expected to down-regulate theseinflammatory stimuli. Thus, uroguanylin-type agonists should be usefulin the prevention and treatment of inflammatory diseases of the lung(e.g., asthma), bowel (e.g., ulcerative colitis and Crohn's disease),pancreas and other organs.

Overall, it may be concluded that agonists of guanylate cyclase receptorsuch as uroguanylin have potential therapeutic value in the treatment ofa wide variety of inflammatory conditions, cancer (particularly coloncancer) and as anti-metastatic agents. The development of new agonistsis therefore of substantial clinical importance.

SUMMARY OF THE INVENTION

The present invention is based upon the development of new agonists ofguanylate cyclase receptor, and new uses of naturally occurringagonists. The agonists are analogs of uroguanylin, many of which havesuperior properties either in terms of improved receptor activation,stability, activity at low pH or reduced adverse effects. The peptidesmay be used to treat any condition that responds to enhancedintracellular levels of cGMP. Intracellular levels of cGMP can beincreased by enhancing intracellular production of cGMP and/or byinhibition of its degradation by cGMP-specific phosphodiesterases. Amongthe specific conditions that can be treated or prevented areinflammatory conditions, cancer, polyps, and metastasis.

In its first aspect, the present invention is directed to a peptideconsisting essentially of the amino acid sequence of any one of SEQ IDNOs:2-21 and to therapeutic compositions which contain these peptides.The term “consisting essentially of” includes peptides that areidentical to a recited sequence identification number and othersequences that do not differ substantially in terms of either structureor function. For the purpose of the present application, a peptidediffers substantially if its structure varies by more than three aminoacids from a peptide of SEQ ID NOs:2-21 or if its activation of cellularcGMP production is reduced or enhanced by more than 50%. Preferably,substantially similar peptides should differ by no more than two aminoacids and not differ by more than about 25% with respect to activatingcGMP production. The most preferred peptide is a bicycle having thesequence of SEQ ID NO:20.

The peptides may be in a pharmaceutical composition in unit dose form,together with one or more pharmaceutically acceptable excipients. Theterm “unit dose form” refers to a single drug delivery entity, e.g., atablet, capsule, solution or inhalation formulation. The amount ofpeptide present should be sufficient to have a positive therapeuticeffect when administered to a patient (typically, between 100 μg and 3g). What constitutes a “positive therapeutic effect” will depend uponthe particular condition being treated and will include any significantimprovement in a condition readily recognized by one of skill in theart. For example, it may constitute a reduction in inflammation, ashrinkage of polyps or tumors, a reduction in metastatic lesions, etc.

The invention also encompasses combination therapy utilizing a guanylatecyclase receptor agonist administered either alone or together with aninhibitor of cGMP-dependent phosphodiesterase, an anti-inflammatoryagent or an anticancer agent. These agents should be present in amountsknown in the art to be therapeutically effective when administered to apatient. Anti-neoplastic agents may include alkylating agents,epipodophyllotoxins, nitrosoureas, antimetabolites, vinca alkaloids,anthracycline antibiotics, nitrogen mustard agents, and the like.Particular anti-neoplastic agents may include tamoxifen, TAXOL™,etoposide and 5-fluorouracil. Antiviral and monoclonal antibodytherapies may be combined with chemotherapeutic compositions comprisingat least one guanylate cyclase receptor agonist in devising a treatmentregimen tailored to a patient's specific needs.

In another aspect, the invention is directed to a method for preventing,treating or retarding the onset of cancer, particularly cancer ofepithelial cells, or polyps in a subject by administering a compositioncomprising an effective amount of a guanylate cyclase receptor agonist,preferably a synthetic guanylate cyclase receptor agonist. The term“effective amount” refers to sufficient agonist to measurably increaseintracellular levels of cGMP. The term “synthetic” refers to a peptidecreated to bind a guanylate cyclase receptor, but containing certainamino acid sequence substitutions not present in known endogenousguanylate cyclase agonists, such as uroguanylin. The agonist should be apeptide selected from those defined by SEQ ID NOs:2-21 and which arelisted in Tables 2 and 3. Also included in the invention are methods oftreating primary cancers, other than primary colon cancer, byadministering an effective dosage of a peptide selected from the groupconsisting of: uroguanylin; guanylin; and E. coli ST peptide. Any knownform of uroguanylin or guanylin can be used for this purpose, althoughthe human peptides are preferred.

The invention also includes methods of preventing or treating tumormetastasis from a primary tumor mass. Metastatic tumor cells havingguanylate cyclase receptors may be targeted by peptides generatedaccording to the invention. In a preferred embodiment, the targetedreceptor is found on cells of gastrointestinal (GI) cancers and onmetastasized cells derived from those cancers. Such receptors aretypically transmembrane proteins with an extracellular ligand-bindingdomain, a membrane-spanning domain, and an intracellular domain withguanylate cyclase activity. Although the invention is not bound by anyparticular mechanism of action, it is believed that the peptides willact by binding to these cellular receptors and inducing apoptosis.Metastatic tumors may also be treated by administering any known form ofuroguanylin or guanylin (preferably human) or by administering E. coliST peptide.

Peptides may be administered either alone or together with one or moreinhibitors of cGMP dependent phosphodiesterase. Examples of cGMPdependent phosphodiesterase inhibitors include suldinac sulfone,zaprinast, and motapizone. Treatable forms of cancer include breastcancer, colorectal cancer, lung cancer, ovarian cancer, pancreaticcancer, prostate cancer, renal cancer, and testicular cancer. Coloncarcinogenesis may be prevented by inhibiting pre-cancerous colorectalpolyp development via administration of a composition according to theinvention. It is believed that the peptides should be especiallyeffective with respect to the treatment of colon cancer and inpreventing the metastasis of colon tumors.

In another aspect, the invention is directed to a method for treating,preventing, or retarding the onset of organ inflammation (e.g.,inflammation associated with the GI tract, asthma, nephritis, hepatitis,pancreatitis, bronchitis, or cystic fibrosis) of a subject byadministering a composition comprising an agonist of a guanylate cyclasereceptor that enhances intracellular production of cGMP. Preferredpeptide agonists are selected from the group defined by SEQ ID NOs:2-21shown in Tables 2 and 3, or uroguanylin, or guanylin, or E. coli STpeptide. These peptides may optionally be administered with one or moreinhibitors of cGMP dependent phosphodiesterase, e.g., suldinac sulfone,zaprinast, or motapizone. In a preferred embodiment, the invention isdirected to a method of treating an inflammatory disorder in a mammaliangastrointestinal tract. The inflammatory disorder may be classified asan inflammatory bowel disease, and more particularly may be Crohn'sdisease or ulcerative colitis. Administration may be enteric, and employformulations tailored to target enterocytes.

In a broader sense, the invention includes methods of inducing apoptosisin a patient by administering an effective amount of a peptide havingthe sequence of any one of SEQ ID NO:2-SEQ ID NO:21, or uroguanylin, orguanylin or E. coli ST peptide. An “effective amount” of peptide, inthis sense, refers to an amount sufficient to increase apoptosis in atarget tissue. For example, sufficient peptide may be given to induce anincreased rate of cell death in a neoplastic growth.

The most preferred peptide for use in the methods described above is thepeptide defined by SEQ ID NO:20. The sequence is as follows (see alsoTable 3):

Asn¹ Asp² Glu³ Cys⁴ Glu⁵ Leu⁶ Cys⁷ Val⁸ Asn⁹ Val¹⁰ Ala¹¹ Cys¹² Thr¹³ Gly¹⁴ Cys¹⁵ Leu¹⁶                *             **                          *                **and wherein there is one disulfide linkage between the cysteine atposition 4 and the cysteine at position 12; and a second disulfidelinkage between the cysteine at position 7 and the cysteine at position15 (SEQ ID NO:20). This peptide has been found to have enhancedbiological activity as an agonist of cGMP production due to its enhancedbinding constant for the guanylate cyclase receptor, and is superior touroguanylin with regard to temperature and protease stability and withregard to its biological activity at the physiologically favorable pHrange (pH 6 to 7) in the large intestine.

The guanylate cyclase receptor agonists used in the methods describedabove may be administered either orally, systemically or locally. Dosageforms include preparations for inhalation or injection, solutions,suspensions, emulsions, tablets, capsules, topical salves and lotions,transdermal compositions, other known peptide formulations and pegylatedpeptide analogs. An effective dosage of the composition will typicallybe between about 1 .mu.g and about 10 mg per kilogram body weight,preferably between about 10 μg to 5 mg of the compound per kilogram bodyweight. Adjustments in dosage will be made using methods that areroutine in the art and will be based upon the particular compositionbeing used and clinical considerations. Agonists may be administered aseither the sole active agent or in combination with other drugs, e.g.,an inhibitor of cGMP-dependent phosphodiesterase. In all cases,additional drugs should be administered at a dosage that istherapeutically effective using the existing art as a guide. Drugs maybe administered in a single composition or sequentially.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon several concepts. The first is thatthere is a cGMP-dependent mechanism which regulates the balance betweencellular proliferation and apoptosis and that a reduction in cGMPlevels, due to a deficiency of uroguanylin/guanylin and/or due to theactivation of cGMP-specific phosphodiesterases, is an early and criticalstep in neoplastic transformation. A second concept is that the releaseof arachidonic acid from membrane phospholipids, which leads to theactivation of cPLA₂, COX-2 and possibly 5-lipoxygenase during theprocess of inflammation, is down-regulated by a cGMP-dependentmechanism, leading to reduced levels of prostaglandins and leukotrienes,and that increasing intracellular levels of cGMP may therefore producean anti-inflammatory response. In addition, a cGMP-dependent mechanism,is thought to be involved in the control of proinflammatory processes.Therefore, elevating intracellular levels of cGMP may be used as a meansof treating and controlling inflammatory bowel diseases such asulcerative colitis and Crohn's disease and other organ inflammation(e.g., associated with asthma, nephritis, hepatitis, pancreatitis,bronchitis, cystic fibrosis).

Without intending to be bound by any theory, it is envisioned that iontransport across the plasma membrane may prove to be an importantregulator of the balance between cell proliferation and apoptosis thatwill be affected by compositions altering cGMP concentrations.Uroguanylin has been shown to stimulate K⁺ efflux, Ca⁺⁺ influx and watertransport in the gastrointestinal tract (3). Moreover, atrialnatriuretic peptide (ANP), a peptide that also binds to a specificguanylate cyclase receptor, has also been shown to induce apoptosis inrat mesangial cells, and to induce apoptosis in cardiac myocytes by acGMP mechanism (26-29). It is believed that binding of the presentagonists to a guanylate cyclase receptor stimulates production of cGMP.This ligand-receptor interaction, via activation of a cascade ofcGMP-dependent protein kinases and CFTR, is then expected to induceapoptosis in target cells. Therefore, administration of the novelpeptides defined by SEQ ID NOs:2-21, as shown in Tables 2 and 3, oruroguanylin, or guanylin or E. coli ST peptide is expected to eliminateor, at least retard, the onset of inflammatory diseases of the GI tractand general organ inflammation (e.g., asthma, nephritis, hepatitis,pancreatitis, bronchitis, cystic fibrosis).

In another aspect, the invention is directed to a method for preventing,treating or retarding the onset of cancer, particularly cancer ofepithelial cells, in a subject by administering a composition comprisingan effective amount of a guanylate cyclase receptor agonist, preferablya synthetic a guanylate cyclase receptor agonist. The term “effectiveamount” refers to sufficient agonist to measurably increaseintracellular levels of cGMP. The term “synthetic” refers to a peptidecreated to bind a guanylate cyclase receptor, but containing certainamino acid sequence substitutions not present in known endogenousguanylate cyclase agonists, such as uroguanylin. The agonist should be apeptide selected from those defined by SEQ ID NOs:2-21 and which arelisted in Tables 2 and 3. Also included in the invention are methods oftreating primary and metastatic cancers, other than primary coloncancer, by administering an effective dosage of a peptide selected fromthe group consisting of: uroguanylin; guanylin; and E. coli ST peptide.Any known form of uroguanylin or guanylin can be used for this purpose,although the human peptides are preferred.

The cGMP-dependent mechanism that regulates the balance between cellularproliferation and apoptosis in metastatic tumor cells may serve as amechanism for targeting and treating metastatic tumors. The liver is themost common site of metastasis from a primary colorectal cancer. Towardlater stages of disease, colorectal metastatic cells may also invadeother parts of the body. It is important to note that metastatic cellsoriginating from the primary site in the gastrointestinal tracttypically continue to express guanylate cyclase receptors and therefore,these cells should be sensitive to apoptosis therapy mediated byintestinal guanylate cyclase receptors. Peptides having uroguanylinactivity, when used either alone or in combination with specificinhibitors of cGMP-phosphodiesterase, also retard the onset ofcarcinogenesis in gut epithelium by restoring a healthy balance betweencell proliferation and apoptosis via a cGMP-mediated mechanism.

As used herein, the term “guanylate cyclase receptor” refers to theclass of guanylate cyclase receptors on any cell type to which theinventive agonist peptides or natural agonists described herein bind.

As used herein, the term “guanylate cyclase receptor-agonist” refers topeptides and/or other compounds that bind to a guanylate cyclasereceptor and stimulate cGMP production. The term also includes allpeptides that have amino acid sequences substantially equivalent to atleast a portion of the binding domain comprising amino acid residues3-15 of SEQ ID NO: 1. This term also covers fragments and pro-peptidesthat bind to guanylate cyclase receptor and stimulate cGMP production.The term “substantially equivalent” refers to a peptide that has anamino acid sequence equivalent to that of the binding domain wherecertain residues may be deleted or replaced with other amino acidswithout impairing the peptide's ability to bind to a guanylate cyclasereceptor and stimulate cGMP production.

Strategy and Design of Novel Guanylate Cyclase Receptor Agonists

Uroguanylin is a peptide secreted by the goblet and other epithelialcells lining the gastrointestinal mucosa as pro-uroguanylin, afunctionally inactive form. The human pro-peptide is subsequentlyconverted to the functionally active 16 amino acid peptide set forth inSEQ ID NO:1 (human uroguanylin sequence, see Table 2) in the lumen ofthe intestine by endogenous proteases. Since uroguanylin is aheat-resistant, acid-resistant, and proteolysis-resistant peptide, oralor systemic administration of this peptide and/or other peptides similarto the functionally active 16 amino acid peptide sequence of SEQ ID NO:1may be effectively employed in treatment methods.

Peptides similar to, but distinct from, uroguanylin are described below,including some which produce superior cGMP enhancing properties and/orother beneficial characteristics (e.g., improved temperature stability,enhanced protease stability, or superior activity at preferred pH's)compared to previously known uroguanylin peptides. The peptides may beused to inhibit GI inflammation and for treating or preventing the onsetof polyp formation associated with gut inflammation. Epithelial tissuessusceptible to cancer cell formation may also be treated. The guanylatecyclase receptor agonists described have the amino acid sequences shownin Tables 2 and 3. The “binding domain” for agonist-receptor interactionincludes the amino acid residues from 3-15 of SEQ ID NO:1.

Molecular modeling was applied to the design of novel guanylate cyclasereceptor agonists using methods detailed in (30). It consisted of energycalculations for three compounds known to interact with guanylatecyclase receptors, namely for human uroguanylin, bicyclo [4,12;7,15]Asn¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶(UG, SEQ ID NO:1); human guanylin, bicyclo [4,12;7,15]Pro¹-Gly²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Tyr⁹-Ala¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-¹⁵Cys(GU, SEQ ID NO:22); and E. coli small heat-stable enterotoxin, tricyclo[6,10; 7,15; 11-18]Asn¹-Ser²-Ser³-Asn⁴-Tyr⁵-Cys⁶-Cys⁷-Glu⁸-Leu⁹-Cys¹⁰-Cys¹¹-Asn¹²-Pro¹³-Ala¹⁴-Cys¹⁵-Thr¹⁶-Gly¹⁷-Cys¹⁸-Tyr¹⁹(ST, SEQ ID NO:23). Geometrical comparisons of all possible low-energyconformations for these three compounds were used to reveal the common3D structures that served as the “templates” for the bioactiveconformation, i.e., for the conformation presumably adopted by GU, UGand ST during interaction with receptor. It allowed designing novelanalogs with significantly increased conformational population of thebioactive conformation at the expense of other low-energy conformationsby selecting individual substitutions for various amino acid residues.

Energy calculations were performed by use of build-up procedures (30).The ECEPP/2 potential field (31,32) was used assuming rigid valencegeometry with planar trans-peptide bonds, including that for Pro¹³ inST. The ω angle in Pro¹³ was allowed to vary. Aliphatic and aromatichydrogens were generally included in united atomic centers of CH_(n)type; H^(α)-atoms and amide hydrogens were described explicitly.

The main calculation scheme involved several successive steps. First,the sequences of the two monocyclic model fragments (three fragments forST), Ac-cyclo (Cys^(i)- . . . -Cys^(i))-NMe, were considered, where allresidues except Cys, Gly and Pro were replaced by alanines; the i and jvalues corresponded to the sequences of GU, UG and ST. At this step, allpossible combinations of local minima for the peptide backbone for eachamino acid residue were considered, i.e., the minima in the Ramachandranmap of E, F, C, D, A and A* types (according to the notation in (33))for the Ala residue; of E*, F*, C*, D*, A, E, F, C D and A * types forthe Gly residue; and of F, C and A types for Pro. For each backboneconformation, one optimal possibility to close a cycle employing theparabolic potential functions, intrinsic to the ECEPP force field, wasfound by checking an energy profile of rotation around the dihedralangle χ1 for the D-Cys residue.

Totally, as many as ca. 180,000 conformations for each of the cyclicmoieties were considered. Then, the conformers satisfying theE−E_(min)<ΔE=15 kcal/mol criterion and differing by more than 40° in atleast one value of any backbone dihedral angle were selected (from ca.3,000 to 8,000 conformations for different model fragments). At the nextstep, the selected conformations of the matching monocyclic fragmentswere overlapped to create possible conformations of the bicyclic modelfragments (the tricyclic fragments in the case of ST). Typically, thisprocedure yielded ca. 20,000-30,000 conformations. All theseconformations were submitted for a new cycle of energy calculations,which resulted in 191 conformations satisfying the E−E_(min)<ΔE=20kcal/mol criterion for the ST model fragment and in 6,965 conformationssatisfying the same criterion for the GU/UG model fragment. After that,the missing side chains in the model fragments were restored, and energycalculations were performed again, the dihedral angle values of sidechain groups (except the χ1 angle for the Cys residues) and of theterminal groups of the backbone being optimized before energyminimization to achieve their most favorable spatial arrangements,employing an algorithm previously described (34). For the UG 4-15fragment, 632 conformations satisfied the criterion of ΔE=20 kcal/mol;164 of them satisfied the more stringent criterion of ΔE=12 kcal/mol,which corresponds to the accepted criterion of 1 kcal/mol/residue (30).Subsequent elongation of the UG 4-15 fragment to 3-16, and then to theentire UG molecule was performed by the same build-up procedure.Finally, 31 backbone conformations of UG were found as satisfying thecriterion of ΔE=16 kcal/mol.

Geometrical comparison of conformers was performed in the followingmanner. The best fit in the superposition for the atomic centers in apair of conformers was assessed to check the level of geometricalsimilarity between the two conformers, according to (35). The criterionfor geometrical similarity was the rms value, which was calculated for apair of conformations A and B as follows:rms=(1/N)Σ^(N) i=1 [(x ^(A) i−x ^(B) i)²+(y ^(A) i−y ^(B) i)²+(z ^(A)i−z ^(B) i)²]^(1/2),where N is the number of the C^(α)-atom pairs chosen for superposition,and x, y and z are the Cartesian coordinates. By the criterion ofgeometrical similarity of rms<2.0 Å, low-energy conformations of therigid conformational fragment UG 4-15 fell into seven conformationalfamilies. One of them consists of the same six conformers that aresimilar both to 1UYA and 1ETN; this family contains also thelowest-energy conformer of UG. (1UYA and 1ETN are the experimentallydefined 3D structures of UG and ST, respectively, which are known topossess high biological activity (36,37); the 3D structures wereavailable in the Protein Data Bank.)

TABLE 1 The values of dihedral angles (in degrees) for peptide backbonein the “template” conformation of UG Conformer's # Residue Angle 1 3 922 25 27 Cys⁴ Ψ −37 −41 −40 −55 −38 −54 Glu⁵ φ −71 −67 −72 −69 −68 −70 ψ−50 −47 −48 −33 −43 −22 Leu⁶ φ −86 −86 −85 −81 −88 −91 ψ 163 165 160 153160 156 Cys⁷ φ −79 −82 −79 −83 −79 −81 ψ 74 68 78 67 75 72 Val⁸ φ −120−114 −126 −124 −125 −128 ψ −65 −57 −62 −55 −60 −64 Asn⁹ φ −83 −95 −82−88 −89 −82 Ψ 119 113 134 118 111 116 Val¹⁰ φ −84 −82 −97 −90 −82 −82 ψ−21 −13 −16 −4 −15 −16 Ala¹¹ φ −79 −86 −87 −89 −85 −80 ψ −32 −21 −35 −35−18 −27 Cys¹² φ −86 −92 −78 −79 −95 −90 ψ −52 −53 −55 −57 −53 −54 Thr¹³φ −129 −121 −127 −119 −118 −130 ψ 111 153 141 155 141 119 Gly¹⁴ φ −64−78 −78 −80 −78 −68 ψ 83 64 68 62 67 78 Cys¹⁵ φ −139 −160 −150 −156 −78−131

The dihedral angles Φ and Ψ, values that determine the overall 3D shapeof this UG fragment, are similar (Table 1). It allowed performingpreliminary design of new analogs aimed at stabilizing this particularfamily of conformations employing the known local conformationallimitations imposed by various types of amino acids.

For instance, it is known that Gly is more conformationally flexiblecompared to any other L-amino acid residue, since Gly may adoptconformations with any of the four combinations of signs for Φ and Ψ,i.e., −,+; −,−; +,+; and +,−. The last combination is stericallyforbidden for the L-amino acids, as Ala. Therefore, substitution ofGly¹⁴ for Ala¹⁴ should limit conformational flexibility in position 14preserving the conformations described in Table 1. Also, substitutionfor Aib (α-Me-Ala, di-α-methyl-alanine) should limit the localconformational flexibility by two regions only, namely for −,− and +,+,the first one being compatible with conformers of Ala¹¹ in Table 1.Therefore, one more desirable substitution is Aib¹¹. In Pro, the Φ valueis fixed at −75°; this residue is also similar to valine by itshydrophobic properties. Therefore, Val¹⁰ may be replaced by Pro¹⁰, whichadds more local conformational constraints to the UG conformers inTable 1. Replacement by Pro also requires that the preceding residuepossesses only positive Ψ values; Asn⁹ in Table 1 fulfills thisrequirement. The Pro residue already exists in the correspondingposition of ST. All suggested substitutions within SEQ ID NO:1 shownbelow (e.g., Pro¹⁰, Aib¹¹ or Ala¹⁴) do not change the chemical nature ofthe non-aliphatic amino acids (such as Asn, Asp or Thr), which may beimportant for the actual interaction with receptor. The formersubstitutions should lead only to conformational limitations shiftingconformational equilibrium in UG towards the suggested “template” 3-Dshape.

Based on the 3D structures defined in Table 1, a three-dimensionalpharmacophore for uroguanylin was defined, enabling the determination ofdistances between functional groups of uroguanylin thought to directlyinteract with the receptor. Those groups thought to directly interactwith the receptor are side groups of residues in positions 3, 5, 9 and13 of the backbone sequence. Preferably, the residues are Glu3, Glu5,Asn9, and Thr13, as shown in SEQ ID NO:2 and SEQ ID NO:20. Thus, a threedimensional pharmacophore of uroguanylin is described in which thespatial arrangement of the four side chains of the residues at positions3, 5, 9 and 13 may be created such that the distances between these sidechains enable optional biological activity. Those distances (measured asdistances between C.beta. atoms of corresponding residues) are asfollows: from 5.7 to 7.6 Å for the 3-5 distance, from 4.0 to 6.0 Å for3-9; from 7.7 to 8.3 Å for 3-13, from 9.4 to 9.5 Å for 5-9, from 9.4 to9.5 Å for 5-13, and from 5.8 to 6.3 Å for 9-13.

The distances above depend only on conformations of the peptidebackbone. In some cases, however, conformations of side chainsthemselves are also important. For instance, calculations showed thatthere is no conformational difference between the backbones of UG(SP301), [Glu²]-UG (SP303), [Glu³]-UG (SP304) and [Glu², Glu³]-UG(SP302) in terms of their low-energy conformations. However, there is adistinct difference in the spatial positions of the β-carboxyls of Aspand γ-carboxyls of Glu in position 3. Namely, γ-carboxyls of the Gluresidues in position 3 are clearly stretched “outwards” of the bulk ofthe molecules farther than the corresponding β-carboxyls of the Aspresidues. The above observation strongly suggests that the negativelycharged carboxyl group of the side chain in position 3 specificallyinteracts with a positively charged binding site on the receptor;therefore, analogs containing Glu³ instead of Asp³ should be moreactive. At the same time, to ensure efficiency of this particularinteraction, an entire system of the long-range electrostaticinteractions between ligand and receptor should be well balanced. Sincethe Glu² side chain presents more conformational possibilities comparedto the Asp² side chain, this balance may be slightly changed in SP302(double substitution of Asp's for Glu's) compared to SP304 (singlesubstitution of Asp³ for Glu³).

Compounds capable of adopting low-energy conformations described inTable 1 are listed in Table 2. All compounds are [4,12; 7,15] bicycles.

TABLE 2 1. Parent compound, uroguanylin SEQ ID NO:1Asn¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶ 2. Compounds withoutmodifications of cysteines: Common sequence (SEQ ID NO:2):Asn¹-Xaa²-Xaa³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Thr¹³-Xaa¹⁴-Cys¹⁵-Leu¹⁶ where Xaa² = Asp, Glu;Xaa³ = Asp, Glu with the exception that Xaa² and Xaa³ are not both Aspin same molecule And where Xaa¹⁰ = Val, Pro; Xaa¹¹ = Ala, Aib;Xaa¹⁴ = Gly, Ala 3. Compounds with mercaptoproline (Mpt) substi- tutedfor cysteine in position 7: Common sequence (SEQ ID NO:3):Asn¹-Xaa²-Xaa³-Cys⁴-Glu⁵-Leu⁶-Xaa⁷-Val⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Thr¹³-Xaa¹⁴-Cys¹⁵-Leu¹⁶ where Xaa² = Asp, Glu;Xaa³ = Asp, Glu where Xaa¹⁰ = Val, Pro; Xaa¹¹ = Ala, Aib; Xaa¹⁴ = Gly,Ala 4. Compounds with penicillamines (β, β-dimethyl- cysteines, Pen)substituted for cysteines: Common sequence (SEQ ID NO:4):Asn¹-Xaa²-Xaa³-Xaa⁴-Glu⁵-Leu⁶-Xaa⁷-Val⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Xaa¹²-Thr¹³-Xaa¹⁴-Xaa¹⁵-Leu¹⁶ where Xaa² = Asp, Glu;Xaa³ = Asp, Glu where Xaa¹⁰ = Val, Pro; Xaa¹¹ = Ala, Aib; Xaa¹⁴ = Gly,Ala and Xaa⁴, Xaa⁷, Xaa¹², Xaa¹⁵ are either Cys or Pen (except not allare Cys in the same conformer) 5. Compounds with lactam bridgessubstituted for disulfide bridges: Common sequence (SEQ ID NO:5):Asn¹-Xaa²-Xaa³-Xaa⁴-Glu⁵-Leu⁶-Xaa⁷-Val⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Xaa¹²-Thr¹³-Xaa¹⁴-Xaa¹⁵-Leu¹⁶ where Xaa² = Asp, Glu; Xaa³= Asp, Glu where Xaa¹⁰ = Val, Pro; Xaa¹¹ = Ala, Aib; Xaa¹⁴ = Gly, Alaand all combinations of the following (Dpr is diaminopropionic acid):Xaa⁴ is either Asp or Glu, and Xaa¹² is Dpr; Xaa⁷ is either Cys or Pen;Xaa¹⁵ is either Cys or Pen; or: Xaa⁷ is Dpr and Xaa¹⁵ is either Asp orGlu; Xaa⁷ is either Asp or Glu, and Xaa¹⁵ is Dpr; Xaa⁴ is either Cys orPen; Xaa¹² is either Cys or Pen;

Some of the peptides shown in Table 2 contain 16 amino acid residues inwhich cysteine residues form disulfide bridges between Cys⁴ and Cys¹²,and Cys⁷ and Cys¹⁵, respectively. These peptides differ from the peptidesequences described in WO 01/25266, and are designed on the basis ofpeptide conformation and energy calculations.

In addition, peptides, varying in length from 13 to 16 amino acids,shown in Table 3, are designed, based on energy calculations andthree-dimensional structures, to promote stabilization of thebiologically active conformer and minimize or eliminate interconversionto biologically inactive conformers. These peptides are also designed topromote stability against proteolysis and higher temperatures. Thedesign of these peptides involves modifications of amino acid residuesthat contain ionic charges at lower pH values, such as glutamic andaspartic acids.

TABLE 3 SEQ ID NO:6 X1 Glu Glu Cys X2  X3  Cys X4  Asn X5  X6  CysX7  X8  Cys X9 SEQ ID NO:7 X1 Glu Asp Cys X2  X3  Cys X4  AsnX5  X6  Cys X7  X8  Cys X9 SEQ ID NO:8 X1 Asp Glu Cys X2  X3  CysX4  Asn X5  X6  Cys X7  X8  Cys X9 SEQ ID NO:9 X1 Asp Asp CysX2  X3  Cys X4  Tyr X5  X6  Cys X7  X8  Cys X9 SEQ ID NO:10 X1 Glu GluCys X2  X3  Cys X4  Tyr X5  X6  Cys X7  X8  Cys X9 SEQ ID NO:11 X1 AspGlu Cys X2  X3  Cys X4  Tyr X5  X6  Cys X7  X8  Cys X9 SEQ ID NO:12 X1Glu Asp Cys X2  X3  Cys X4  Tyr X5  X6  Cys X7  X8  Cys X9 SEQ ID NO:13X1 Asp Asp Cys X2  X3  Cys X4  Gln X5  X6  Cys X7  X8  Cys X9 SEQ IDNO:14 X1 Glu Glu Cys X2  X3  Cys X4  Gln X5  X6  Cys X7  X8  Cys X9 SEQID NO:15 X1 Asp Glu Cys X2  X3  Cys X4  Gln X5  X6  Cys X7  X8  Cys X9SEQ ID NO:16 X1 Glu Asp Cys X2  X3  Cys X4  Gln X5  X6  Cys X7  X8  CysX9 SEQ ID NO:17     Glu Cys X2  X3  Cys X4  Asn X5  X6  Cys X7  X8  CysX9 SEQ ID NO:18     Glu Cys X2  X3  Cys X4  Asn X5  X6  Cys X7  X8  CysSEQ ID NO:19 X1  Glu Cys X2  X3  Cys X4  Asn X5  X6  Cys X7  X8  Cys X9 1 2   3   4   5   6   7   8   9   10  11  12  13  14  15  16 SEQ IDNO:20 Asn Asp Gln Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys LeuSEQ ID NO:21 Glu Cys Gln Leu Cys Val Asn Val Ala Cys Thr Gly Cys LeuX1 to X9 can be any amino acid. The disulfide bridges are formed betweenCys residues at 4 and 12 and between 7 and 15, respectively. SEQ IDNO:18 represents the minimum length requirement for these peptides tobind a guanylate cyclase receptor.

Pharmaceutical Compositions and Formulations

The guanylate cyclase receptor agonists of the present invention (Table2; SEQ ID NOs:2-5 and Table 3; SEQ ID NOs:6-21), as well as uroguanylin,guanylin and/or bacterial enterotoxin ST, may be combined or formulatedwith various excipients, vehicles or adjuvants for oral, local orsystemic administration. Peptide compositions may be administered insolutions, powders, suspensions, emulsions, tablets, capsules,transdermal patches, ointments, or other formulations. Formulations anddosage forms may be made using methods well known in the art (see, e.g.,Remington's Pharmaceutical Sciences, 16^(th) ed., A. Oslo ed., Easton,Pa. (1980)).

Inhibitors of cGMP-dependent phosphodiesterase may be small molecules,peptides, proteins or other compounds that specifically prevent thedegradation of cGMP. Inhibitory compounds include suldinac sulfone,zaprinast, motapizone and other compounds that block the enzymaticactivity of cGMP-specific phosphodiesterases. One or more of thesecompounds may be combined with a guanylate cyclase receptor agonistexemplified in SEQ ID NOs:2-21, uroguanylin, guanylin and E. Coli STpeptide.

The selection of carriers (e.g., phosphate-buffered saline or PBS) andother components suitable for use in compositions is well within thelevel of skill in this art. In addition to containing one or moreguanylate cyclase receptor agonists, such compositions may incorporatepharmaceutically acceptable carriers and other ingredients known tofacilitate administration and/or enhance uptake. Other formulations,such as microspheres, nanoparticles, liposomes, pegylated protein orpeptide, and immunologically-based systems may also be used. Examplesinclude formulations employing polymers (e.g., 20% w/v polyethyleneglycol) or cellulose, or enteric formulations and pegylated peptideanalogs for increasing systemic half-life and stability.

Treatment Methods

The term “treatment” refers to reducing or alleviating symptoms in asubject, preventing symptoms from worsening or progressing, orpreventing disease development. For a given subject, improvement in asymptom, its worsening, regression, or progression may be determined byany objective or subjective measure typically employed by one of skillin the art. Efficacy of the treatment in the case of cancer may bemeasured as an improvement in morbidity or mortality (e.g., lengtheningof the survival curve for a selected population). Thus, effectivetreatment would include therapy of existing disease, control of diseaseby slowing or stopping its progression, prevention of diseaseoccurrence, reduction in the number or severity of symptoms, or acombination thereof. The effect may be shown in a controlled study usingone or more statistically significant criteria.

Combination therapy with one or more medical/surgical procedures and/orat least one other chemotherapeutic agent may be practiced with theinvention. Other suitable agents useful in combination therapy includeanti-inflammatory drugs such as, for example, steroids or non-steroidalanti-inflammatory drugs (NSAIDS), such as aspirin and the like.Prophylactic methods for preventing or reducing the incidence of relapseare also considered treatment.

Cancers expected to be responsive to compositions include breast,colorectal, lung, ovarian, pancreatic, prostatic, renal, stomach,bladder, liver, esophageal and testicular carcinoma. Further examples ofdiseases involving cancerous or precancerous tissues that should beresponsive to a therapeutic comprising at least one guanylate cyclasereceptor agonist include: carcinoma (e.g., basal cell, basosquamous,Brown-Pearce, ductal, Ehrlich tumor, in situ, Krebs, Merkel cell, smallor non-small cell lung, oat cell, papillary, bronchiolar, squamous cell,transitional cell, Walker), leukemia (e.g., B-cell, T-cell, HTLV, acuteor chronic lymphocytic, mast cell, myeloid), histiocytoma,histiocytosis, Hodgkin disease, non-Hodgkin lymphoma, plasmacytoma,reticuloendotheliosis, adenoma, adeno-carcinoma, adenofibroma,adenolymphoma, ameloblastoma, angiokeratoma, angiolymphoid hyperplasiawith eosinophilia, sclerosing angioma, angiomatosis, apudoma,branchioma, malignant carcinoid syndrome, carcinoid heart disease,carcinosarcoma, cementoma, cholangioma, cholesteatoma, chondrosarcoma,chondroblastoma, chondrosarcoma, chordoma, choristoma,craniopharyngioma, chrondroma, cylindroma, cystadenocarcinoma,cystadenoma, cystosarcoma phyllodes, dysgerminoma, ependymoma, Ewingsarcoma, fibroma, fibro-sarcoma, giant cell tumor, ganglioneuroma,glioblastoma, glomangioma, granulosa cell tumor, gynandroblastoma,hamartoma, hemangioendothelioma, hemangioma, hemangio-pericytoma,hemangiosarcoma, hepatoma, islet cell tumor, Kaposi sarcoma, leiomyoma,leiomyosarcoma, leukosarcoma, Leydig cell tumor, lipoma, liposarcoma,lymphangioma, lymphangiomyoma, lymphangiosarcoma, medulloblastoma,meningioma, mesenchymoma, mesonephroma, mesothelioma, myoblastoma,myoma, myosarcoma, myxoma, myxosarcoma, neurilemmoma, neuroma,neuroblastoma, neuroepithelioma, neurofibroma, neurofibromatosis,odontoma, osteoma, osteosarcoma, papilloma, paraganglioma, paragangliomanonchromaffin, pinealoma, rhabdomyoma, rhabdomyosarcoma, Sertoli celltumor, teratoma, theca cell tumor, and other diseases in which cellshave become dysplastic, immortalized, or transformed.

A bolus of the inventive composition may be administered over a shorttime. Once a day is a convenient dosing schedule to treat, inter alia,one of the above-mentioned disease states. Alternatively, the effectivedaily dose may be divided into multiple doses for purposes ofadministration, for example, two to twelve doses per day. The dose levelselected for use will depend on the bioavailability, activity, andstability of the compound, the route of administration, the severity ofthe disease being treated, and the condition of the subject in need oftreatment. It is contemplated that a daily dosage will typically bebetween about 10 μg and about 2 mg (e.g., about 100 μg to 1 mg) of thecompound per kilogram body weight. The amount of compound administeredis dependent upon factors known to a person skilled in this art such as,for example, chemical properties of the compound, route ofadministration, location and type of cancer, and the like.

The subject mammal may be any animal or human patient. Thus, bothveterinary and medical treatments are envisioned according to theinvention.

The invention will be further described by the following non-limitingexample.

EXAMPLES

Materials and Methods

Cell Culture: Human T84 colon carcinoma cells were obtained from theAmerican Type Culture Collection at passage 52. Cells were grown in a1:1 mixture of Ham's F-12 medium and Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% fetal bovine serum, 100 U penicillin/ml,and 100 μg/ml streptomycin. The cells were fed fresh medium every thirdday and split at a confluence of approximately 80%.

T84 cell-based assay for determining the intracellular levels of cGMP:Peptide analogs were custom synthesized by Multiple Peptide Systems, SanDiego, Calif., and by Princeton Biomolecules, Langhorne, Pa. Biologicalactivity of the synthetic peptides was assayed as previously reported(15). Briefly, the confluent monolayers of T-84 cells in 24-well plateswere washed twice with 250 μl of DMEM containing 50 mM HEPES (pH 7.4),pre-incubated at 37° C. for 10 min with 250 μl of DMEM containing 50 mMHEPES (pH 7.4) and 1 mM isobutylmethylxanthine (IBMX), followed byincubation with peptide analogs (0.1 nM to 10 μM) for 30 min. The mediumwas aspirated, and the reaction was terminated by the addition of 3%perchloric acid. Following centrifugation, and neutralization with 0.1 NNaOH, the supernatant was used directly for measurements of cGMP usingan ELISA kit (Caymen Chemical, Ann Arbor, Mich.).

Results

Peptides shown in Table 4 were custom synthesized and purified (>95%purity) using a published procedure (38). Peptide analogs were evaluatedin the T84 cell-based assay for their ability to enhance intracellularlevels of cGMP. As shown in Table 4, SP304 (SEQ ID NO:20) gave thegreatest enhancement of intracellular cGMP of all the analogs tested.SP316 (SEQ ID NO:21) was second in effectiveness, whereas the biologicalactivities of SP301, SP302 and SP303 were all somewhat weaker. Thepeptide analogs SP306 and SP310 were not active in this assay. Theseresults indicate that SP304 is the most potent peptide for enhancingcGMP. These results also suggest that the cysteine residue at position 7cannot be substituted with penicillamine as a component of the [7,15]disulfide linkage, and that the Asn residue at position 9 cannot bechanged to a Gln.

TABLE 4 Peptide agonists evaluated for biological activity in the T84cell bioassay. cGMP Level** SEQ ID NO.* Compound Code (pmol/well) 1 SP301 205 6 SP 302 225 7 SP 303 195 20 SP 304 315 14 SP 306  0 4 SP 310  021 SP 316 275 *SEQ ID's for SP301, SP304 and SP316 are the precise aminoacid sequences for these analogs as given in the text. **IntracellularcGMP level observed in T84 cells following treatment with 1 micromolarsolution of the respective peptide agonist for 30 minutes. The valueobserved for SP304 was statistically significant with a p > 0.5.

To examine heat stability, 10 micromolar solutions of peptide analogswere heated at 95° C. for up to 90 minutes. At specific times during thetreatment, samples were tested for their biological activity in the T84cell-based assay. Biological activity of SP301, SP302, SP303 and SP304did not change significantly after 60 minutes of heating. After 90minutes, the activities of SP301, SP302 and SP303 were reduced to about80% of their original values, whereas the biological activity of SP304remained unaltered. This indicates that SP304 is more stable to heatdenaturation compared to the other peptides tested. Based on energycalculations and 3D structure, we expected that the negatively chargedcarboxyl group of the side chain in position 3 of SEQ ID NO:1specifically interacts with a positively charged binding site on thereceptor. In the case where this interaction can be enhanced, analogscontaining Glu³ instead of Asp³ should be more active, as was found tobe the case with SP304. At the same time, to ensure efficiency of thisparticular interaction, an entire system of the long-range electrostaticinteractions between ligand and receptor should be well balanced. Sincethe Glu² side chain presents more conformational possibilities comparedto the Asp² side chain, this balance may be slightly changed in SP302(double substitution of Asp's for Glu's) compared to SP304 (singlesubstitution of Asp³ for Glu³). Indeed, biological activity of SP 304 isthe best amongst the analogs evaluated.

Synthetic peptides SP301, SP302, SP303 and SP304 were also tested fortheir activities at different pH values of the T84 cell-based assay.Whereas all of these peptides showed enhanced intracellular productionof cGMP at pH's ranging from 5 to 7, SP304 showed the greatestenhancement in the range between 6.5 and 7. It is important to note thatthe physiological pH of the large intestine is in a similar range, and,therefore, SP304 would be expected to be especially efficacious forcolon cancer treatment.

We also evaluated peptides used either alone or in combination withinhibitors of cGMP dependent phosphodiesterase (e.g., zaprinast orsulindac sulfone) in T84 cell-based assays for enhancement ofintracellular levels of cGMP. Combinations of an inhibitor of cGMPdependent phosphodiesterase with SP304 displayed a dramatic effect inenhancing cGMP levels in these experiments. Synthetic peptide SP304substantially increased the cGMP level over the level reached in thepresence of either zaprinast or sulindac sulfone alone. Treatment ofwells with SP304 in combination with either Zaprinast or sulindacsulfone resulted in synergistic increases in intracellular cGMP levels.These increases were statistically significant, with p values of <0.5.These data indicate that treatments combining a peptide agonist of aguanylate cyclase receptor with one or more inhibitors of cGMP dependentphosphodiesterase result in a greater than additive increase in cGMPconcentrations.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to those of ordinaryskill in the art that various changes and modifications can be madewithout departing from the spirit and scope of the invention.

REFERENCES

-   1. Currie, et al., Proc. Nat'l Acad. Sci. USA 89:947-951 (1992).-   2. Hamra, et al., Proc. Nat'l Acad. Sci. USA 90:10464-10468 (1993).-   3. Forte, L., Reg. Pept. 81:25-39 (1999).-   4. Schulz, et al., Cell 63:941-948 (1990).-   5. Guba, et al., Gastroenterology 111:1558-1568 (1996).-   6. Joo, et al., Am. J. Physiol. 274:G633-G644 (1998).-   7. Evan, et al., Nature (London) 411:342-348 (2001). 8. Eastwood,    G., J. Clin. Gastroenterol. 14:S29-33 (1992).-   9. Lipkin, M. Arch. Fr. Mal. Appl Dig. 61:691-693 (1972).-   10. Wong, et al., Gut 50:212-217 (2002).-   11. Potten, et al., Stem Cells 15:82-93.-   12. Basoglu, et al., in: Proceedings of the Second FEPS Congress,    Jun. 29-Jul. 4, 1999, Prague, Czech Republic.-   13. Sindic, et al., J. Biol. Chem. Mar. 11, 2002, manuscript    M110627200 (in press).-   14. Zhang, et al., Science 276:1268-1272 (1997).-   15. Shailubhai, et al., Cancer Res. 60:5151-5157 (2000).-   16. Shailubhai, et al., In: Proceedings of the 1999 AACR.NCI.EORTC    International Conference. November 1999, Abstract #0734.-   17. Cohen, et al., Lab. Invest. 78:101-108 (1998).-   18. Sciaky, et al., Genomics 26:427-429 (1995).-   19. Whitaker, et al., Genomics 45:348-354 (1997).-   20. Leister, et al., Cancer Res. 50:7232-7235 (1990).-   21. Cheng, et al., Cell 63:827-834 (1990).-   22. Welsh, et al., Cell 73:1251-1254 (1993).-   23. Weber, et al., Am. J. Physiol. Lung Cell Mol. Physiol.    281(1):L71-78 (2001).-   24. Venkatakrishnan, et al., Am. J. Respir. Cell Mol. Biol.    23(3):396-403 (2000).-   25. Hudson, et al., Free Radic. Biol. Med. 30:1440-1461 (2001).-   26. Bhakdi, et al., Infect. Immun. 57:3512-3519 (1989).-   27. Hughes, et al, J. Biol. Chem. 272:30567-30576 (1997).-   28. Cermak, et al., Pflugers Arch. 43:571-577 (1996).-   29. Wu, et al., J. Biol. Chem. 272:14860-14866 (1997).-   30. Nikiforovich, G., Int. J. Pept. Prot. Res. 44:513-531 (1994).-   31. Dunfield, et al., J. Phys. Chem. 82:2609-2616 (1978).-   32. Nemethy, et al., J. Phys. Chem. 87:1883-1887 (1983).-   33. Zimmerman, et al., Biopolymers 16:811-843 (1977).-   34. Nikiforovich, et al., Biopolymers 31:941-955 (1991).-   35. Nyburg, S., Acta Crystallographica B30 (part 1):251-253 (1974).-   36. Chino, et al., FEBS Letters 421:27-31 (1998).-   37. Schulz, et al., J. Peptide Res. 52:518-525 (1998).-   38. Klodt, et al., J. Peptide Res. 50:222-230 (1997).-   39. Shailubhai, I., Curr. Opin. Drug Discov. Devel. 5:261-268 (2002)

1. A peptide consisting of SEQ ID NO:8, wherein said peptide is a (4,12;7,15) bicycle and wherein said peptide binds a guanylate cyclasereceptor and induces cGMP production.
 2. A pharmaceutical composition inunit dose form comprising a guanylate cyclase receptor agonist peptideconsisting of SEQ ID NO:8 and a pharmaceutically acceptable carrier,wherein said peptide is a (4,12; 7,15) bicycle and wherein said peptidebinds a guanylate cyclase receptor and induces cGMP production.
 3. Thepharmaceutical composition of claim 2, wherein the unit dose form isselected from the group consisting of a tablet, a capsule, a solutionand an inhalation formulation.
 4. The pharmaceutical composition ofclaim 2, further comprising at least one compound selected from thegroup consisting of a cGMP-dependent phosphodiesterase inhibitor, ananti-inflammatory agent, an antiviral agent and an anticancer agent.